Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.

generation by RET 21 and attenuate LPS induction of Il1b mRNA 22 . We investigated the necessity of mitochondrial complex I function for NLRP3 activation by using piericidin A, which decreases OCR and the NAD + /NADH ratio (Extended Data Fig. 3a-c). Piericidin A abolished LPS-induced metabolite changes, including an increase in succinate (Extended Data Fig. 3d,e). Piericidin A did not diminish LPS induction of Il1b, Tnf, or Il10 mRNA after 4 h (Extended Data Fig. 4a-c). Furthermore, piericidin A did not diminish pro-IL-1β protein or pro-caspase-1 protein levels (Extended Data Fig. 4d,e). However, piericidin A did decrease secreted IL-1β protein levels and intracellular cleaved caspase-1 protein levels upon LPS plus ATP stimulation (Extended Data Fig. 4f,g). Piericidin A also decreased secreted IL-1β protein levels in BMDMs treated with the NLRP3 inflammasome activator nigericin (Extended Data Fig.  4h). Importantly, Piericidin A did not diminish LPS induction of secreted TNFα protein levels (Extended Data Fig. 4i) These data suggest that mitochondrial complex I function is not required for the LPS induction of Il1b mRNA but is for caspase-1 activation and production of secreted IL-1β protein.
Reverse electron transport is not required for NLRP3 inflammasome activation. To test the specificity of piericidin A as a mitochondrial complex I inhibitor in our studies, we used BMDMs that express Saccharomyces cerevisiae NADH dehydrogenase (NDI1) 23 . Mammalian mitochondrial complex I transfers electrons from NADH to CoQ while pumping protons across the inner mitochondrial membrane. By contrast, NDI1 transfers electrons from NADH to CoQ but does not pump protons and is unable by itself to generate RET-induced O 2 •− (refs. 23,24 ). Importantly, NDI1 is resistant to piericidin A and other mitochondrial complex I inhibitors 23,25 (Fig. 1a). Thus, treating NDI1-expressing cells with piericidin A allows for NADH oxidation to support downstream electron flow to mitochondrial complexes III, IV and molecular oxygen (respiration), but not complex I-dependent proton pumping or RET-induced O 2 •− production. Recently, we generated a transgenic mouse line that contains a lox-stop-lox-NDI1 targeting construct in the Rosa26 locus 26 . To generate BMDMs that express NDI1, we crossed NDI1 lsl/wt mice with Vav-iCre mice, resulting in mice that express NDI1 in hematopoietic lineages including monocytes, here denoted as NDI1. Mice containing Vav-iCre without the lox-stop-lox-NDI1 are denoted as WT.
As expected, BMDMs generated from NDI1 mice expressed NDI1 mRNA (Fig. 1b). NDI1-expressing BMDMs do not exhibit changes in OCR coupled to ATP production (Fig. 1c). Piericidin A decreased OCR and the NAD + /NADH ratio in WT but not in NDI1-expressing BMDMs (Fig. 1d,e) and decreased RET-generated H 2 O 2 to a similar extent in both WT and NDI1-expressing BMDMs (Fig. 1f). These results indicate that any rescue effects of piericidin A observed in NDI1-expressing cells are independent of RET-generated H 2 O 2 and are due to restoration of NADH oxidation. To understand more broadly any changes in metabolism conferred by the presence of NDI1, we performed metabolomics on BMDMs from WT and NDI1 mice. Metabolites significantly altered in LPS-stimulated WT BMDMs in the presence of piericidin A remained unchanged in NDI1 BMDMs (Fig. 1g). Of note, succinate levels following LPS stimulation were maintained in NDI1 BMDMs in the presence of piericidin A (Fig. 1h).
Next, we determined whether piericidin A inhibition of NLRP3 inflammasome activation was due to inhibition of mitochondrial complex I. NDI1-expressing mice do not have altered IL-1β protein in vivo 2 h post LPS administration (Fig. 2a), indicating that expression of NDI1 itself is not inflammatory. Furthermore, principal component analysis of transcriptional patterns, based on RNA-seq, demonstrated that, both at baseline and in response to LPS, WT and NDI1 BMDMs are largely similar (Extended Data Fig. 5a). However, piericidin A-induced changes in the transcriptional response to LPS treatment were largely abolished by the expression of NDI1 (Extended Data Fig. 5b). Piericidin A attenuated the production of secreted IL-1β protein in WT but not NDI1 BMDMs (Fig. 2b). In contrast, DMM decreased IL-1β protein in both WT and NDI BMDMs, consistent with the proposition that forward electron transport is required to activate the inflammasome (Fig. 2c). Piericidin A also decreased intracellular cleaved caspase-1 protein levels in WT but not NDI1 BMDMs (Fig. 2d). Piericidin A did not decrease intracellular Fig. 1 | NDI1 expression confers resistance to mitochondrial complex I inhibitor piericidin A. a, Schematic of the mitochondrial electron transport chain in WT (top) and NDI1-expressing (bottom) BMDMs during LPS stimulation. Piericidin A inhibition of mitochondrial complex I on electron flow is rescued by NDI1 expression. IMM, inner mitochondrial membrane; RET, reverse electron transport. b, NDI1 mRNA levels (ΔΔC t ) in WT and NDI1 BMDMs (n = 5 WT; n = 12 NDI1). c, Coupled OCR in WT and NDI1 BMDMs (n = 9 for each genotype). d, Basal OCR in WT and NDI1 BMDMs after 1 h treatment with 100 nM or 500 nM piericidin A (n = 13 vehicle for each genotype; n = 9 100 nM piericidin A for each genotype; n = 4 500 nM piericidin A for each genotype). e, NAD + /NADH ratio in WT and NDI1 BMDMs after 4 h treatment with or without LPS (100 ng ml -1 ) in the presence or absence of piericidin A (500 nM) (n = 3 WT LPS + piercidin A; n = 4 all other treatments). f, Rate of H 2 O 2 production in WT and NDI1 BMDMs in the presence of succinate (500 μM) with or without piericidin A treatment (500 nM) (n = 9). g, Heatmap of significantly altered metabolites in WT and NDI1 BMDMs treated with LPS (100 ng ml -1 ) alone, piericidin A alone (500 nM) or both LPS and piericidin A for 4 h. The relative abundance of each metabolite is depicted as z score across rows (red, high; blue, low) (n = 5 for all treatments). h, Arbitrary units of succinate in WT and NDI1 BMDMs with or without LPS (100 ng ml -1 ) and piericidin A (500 nM) for 4 h (n = 5 for all treatments). Data are mean ± s.e.m. *P < 0.05, two-tailed t-test (b, P = 0.0001), ANOVA with Tukey's post hoc test for multiple comparisons (d, *P = 0.0008 WT UT/100 nM, *P = 0.0047 WT UT/500 nM; e, *P = 0.006 WT UT/WT piericidin A, *P = 0.0034 WT LPS/WT LPS + piericidin A; f, *P = 0.0465 WT succinate/WT succinate + piericidin A, *P = 0.0493 NDI1 succinate/NDI1 succinate + piericidin A; h, *P = 0.0478), or ANOVA with Fisher's LSD (g). n indicates number of individual mice. Parts of this figure were created with BioRender.com.     (Fig. 3a). Mitochondrial complex III also pumps protons and is one of the major sites of O 2 •− production. To determine whether mitochondrial complex III is required for inflammasome activation, we treated BMDMs with the inhibitor myxothiazol, which diminished OCR as expected (Extended Data Fig. 6a). As with DMM and   Fig. 6g-i). These results suggest that mitochondrial complex III is also required for NLRP3 inflammasome activation.
To distinguish the role of mitochondrial complex III in electron transport from its ability to generate O 2 •− and proton pump, we adopted an equivalent approach to the use of cells from NDI1 mice by using BMDMs from mice expressing the Ciona intestinalis alternative oxidase (AOX). AOX transfers electrons from CoQH 2 directly to oxygen without proton pumping or O 2 •− production [27][28][29] . In the absence of mitochondrial complex III function, AOX allows mitochondrial complexes I and II to transfer electrons to CoQ, thus regenerating NAD + and FAD without generation of O 2 •− at complex III (ref. 30 ). Moreover, ectopic AOX expression in mammalian cells has been shown to prevent overreduction of the CoQ pool to diminish RET-induced O 2 •− (refs. 21,31 ) (Fig. 3a). In the absence of mitochondrial complex III, AOX expressing cells are able to generate the mitochondrial complex I-dependent proton motive force needed for mitochondrial ATP production, that is, coupled respiration (Fig. 3a) 32 .
To genetically abrogate mitochondrial complex III-and RET-generated O 2
NLRP3 inflammasome activation is not linked to change in mitochondrial membrane potential. Next, we tested whether changes in mitochondrial membrane potential (MPP) was necessary for NLRP3 activation. High or low MMP triggers increase or decrease in ETC-linked superoxide production, respectively 35 . We treated BMDMs with either oligomycin-an inhibitor of mitochondrial complex V (ATP synthase)-or the protonophore carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP). Oligomycin increases both NADH levels and MMP (Extended Data Fig. 7a-c). By contrast, FCCP allows efficient NADH oxidation but decreases MMP (Extended Data Fig. 7a-c). Treatment with oligomycin diminished oxygen consumption, as expected (Extended Data Fig. 8a). Although oligomycin did not significantly diminish the LPS-dependent increase in Il1b mRNA expression or intracellular pro-IL-1β protein levels (Extended Data Fig. 8b,c), it did attenuate the LPS-dependent increase in secreted IL-1β and intracellular cleaved caspase-1 protein  levels in BMDMs treated with extracellular ATP (Extended Data Fig. 8d,e). Oligomycin did not affect intracellular pro-caspase-1 levels (Extended Data Fig. 8f). FCCP did not diminish the LPS-dependent increase in Il1b mRNA expression or intracellular pro-IL-1β protein levels (Extended Data Fig. 8g,h). FCCP did attenuate the LPS-dependent increase in secreted IL-1β and intracellular cleaved caspase-1 protein levels in BMDMs treated with extracellular ATP (Extended Data Fig. 8i, j). FCCP did not affect intracellular pro-caspase-1 levels (Extended Data Fig. 8k). FCCP and oligomycin have distinct effects on the MMP, yet both decrease intracellular cleaved caspase-1 protein levels. Thus, changes in the MMP are not linked to NLRP3 inflammasome activation.
Mitochondrial-generated PCr supports NLRP3 inflammasome activation. To identify one or more common metabolites altered upon inhibition of mitochondrial complexes I, II, III and V and disruption of the MMP, we inspected metabolomics data from cells treated with DMM, piericidin A, myxothiazol, oligomycin or FCCP (Fig. 1g, Extended Data Fig. 1c and Extended Data Fig. 9a,b). PCr was a common metabolite that increased during LPS priming and was diminished by all five inhibitors. The piericidin A-induced decrease in PCr was abrogated by expression of NDI1 (Fig. 1g). PCr is generated from creatine (Cr) and ATP by creatine kinase (CKMT2) in the mitochondria, then released into the cytosol where it is converted back to creatine by cytosolic CKB, transferring the phosphate group to ADP, thus generating cytosolic ATP (Extended Data Fig. 10a). This PCr shuttle provides readily available ATP for energy-consuming processes throughout the rest of the cell 36,37 . To deplete PCr from the cytosol, we treated BMDMs with cyclocreatine (cyCr)-a creatine analog. Cyclocreatine is readily phosphorylated by creatine kinase (CK) to produce phosphocylcocreatine, which is an inefficient donor of phosphate to ADP for ATP generation 38,39 . Treatment of BMDMs with cyCr decreased PCr/Cr levels (Fig. 4a). We measured intracellular ATP levels in BMDMs treated with cyCr to decrease ATP supply via the PCr shuttle or with piericidin A to inhibit mitochondrial complex I. Nigericin administration to LPS-primed BMDMs diminished the level of ATP, which was further decreased by piericidin A or cyCr (Fig. 4b). Cyclocreatine or RNAi against cytosolic CKB also decreased the level of secreted IL-1β in LPS-primed BMDMs treated with extracellular ATP or nigericin (Fig. 5c,d and Extended Data Fig. 9b,c). Cyclocreatine or RNAi against cytosolic CKB decreased intracellular cleaved caspase-1 protein levels without decreasing pro-caspase-1 protein levels ( Fig. 4e-h). The administration of cyCr in vivo diminished LPS induced IL-1β protein in serum (Fig. 5i).
NLRP3 requires ATP hydrolysis for inflammasome activation 40,41 . The widely used NLRP3 inhibitor MCC950 interacts with the Walker B motif within the NLRP3 NACHT domain to prevent ATP hydrolysis 41 . We hypothesized that mitochondrial ETC-generated PCr is required to sustain the cytosolic store of ATP during NLRP3 inflammasome activation. However, LPS stimulation of BMDMs is thought to primarily stimulate glycolysis to sustain ATP levels 42 . Thus, we used extracellular acidification rate (ECAR) to assess glycolytic flux during NLRP3 activation. Nigericin induces cell death in LPS-treated BMDMs after 20 min in a caspase-1 dependent manner (Fig. 5a). Nigericin stimulated ECAR and OCR over 20 min in the presence of the caspase-1 inhibitor VX-765 (Fig. 5b-e). Notably, BMDMs treated with piericidin A during LPS plus nigericin stimulation (which cannot activate the NLRP3 inflammasome) are highly glycolytic (Fig. 5f,g). Thus, glycolysis-generated ATP is not sufficient to support NLRP3 inflammasome activation in the absence of mitochondrial ATP. Importantly, hypoxic cells can generate mitochondrial ATP 43 , which may explain previous work indicating that NLRP3 inflammasome can also be activated under hypoxia (1% O 2 ). Collectively, these data indicate that NLRP3 inflammasome activation depends on mitochondria-derived ATP, initially generated by forward respiratory electron flow and supplied via the PCr shuttle.
NLRP3 inflammasome activation by CL097 requires inhibition of mitochondrial complex I. Typically, activation of the NLRP3 inflammasome requires K + efflux 2 , which occurs upon extracellular ATP or Nigericin administration in LPS-primed BMDMs. However, the NLRP3 inflammasome can also be activated in a K + efflux-independent manner. Notably, K + efflux is dispensable for activation of NLRP3 inflammasome by imiquimod and the related molecule CL097 (ref. 3 ). It has been proposed that these molecules inhibit the quinone oxidoreductases NQO2 and mitochondrial complex I to trigger ROS production, which stimulates NLRP3 inflammasome activation. We tested the necessity of mitochondrial complex I inhibition for CL097-dependent inflammasome activation by using our NDI1-expressing BMDMs. CL097 caused cell death within 20 min in an active caspase-1-dependent manner in LPS-primed BMDMs (Fig. 6a). However, CL097 decreased OCR in the presence of the caspase-1 inhibitor VX-765, indicating that the decrease in OCR was not due to cell death (Fig. 6b). This is consistent with the observation that CL097 decreases OCR in cells lacking inflammasome components 3 . NDI1 expression prevented CL097-or piericidin A-induced decrease in OCR, indicating that CL097 indeed inhibits mitochondrial complex I (Fig. 6c,d). NDI1 expression prevented CL097-dependent secreted IL-1β and intracellular cleaved caspase-1 protein levels in LPS-primed BMDMs without altering intracellular pro-caspase-1 levels ( Fig. 6e-g). Next, we tested whether mitochondrial complex I inhibitor piericidin A or other ETC inhibitors, like CL097, are also sufficient to trigger inflammasome activation in LPS-primed BMDMs. None of the ETC inhibitors increased secreted IL-1β levels (Fig. 6h) indicating that CL097, in addition to inhibiting mitochondrial complex I, has other targets that are necessary for NLRP3 inflammasome activation, perhaps endolysomoal effects 3 . Although piericidin A cannot serve as an inflammasome activator, we tested whether administration of piericidin A or cyclocreatine during LPS priming would diminish CL097 activation of the NLRP3 inflammasome. Indeed, both piericidin A and cyclocreatine administered during LPS priming diminished secreted IL-1β levels and intracellular cleaved caspase-1 protein levels upon CL097 administration (Fig. 6i-n). Thus, mitochondrial-generated ATP to sustain PCr levels during LPS priming is also necessary for CL097 activation of the NLRP3 inflammasome.
CL097 inhibition also triggers ROS production 3 . We tested whether increasing mitochondrial ROS in NDI1-expressing BMDMs, which are resistant to CL097, would rescue NLRP3 inflammasome activation. Antimycin is a well-described generator of mitochondrial superoxide production at complex III (ref. 44 ). Antimycin releases superoxide from mitochondrial complex III  both in the mitochondrial matrix and intermembrane space 44 . By contrast, myxothiazol inhibits mitochondrial superoxide production at complex III 45 . Indeed, antimycin rescued intracellular cleaved caspase-1 (Fig. 7a,b) and secreted IL-1β protein levels ( Fig. 7c) in NDI1-expressing LPS-primed BMDMs treated with CL097. Surprisingly, myxothiazol also increased secreted IL-1β levels in NDI1-expressing LPS-primed BMDMs treated with CL097. Moreover, oligomycin and FCCP, which have opposite effects on MMP and superoxide production, also increased secreted IL-1β levels in NDI1-expressing BMDMs primed with LPS and treated with CL097 (Fig. 7c). FCCP, unlike oligomycin and other ETC inhibitors, allows for efficient NAD + regeneration (Extended Data Fig. 7a) 46 . These results suggest that the rescue effects observed here by ETC inhibitors and FCCP are independent from ROS production or NAD + regeneration. To directly test whether suppressing or scavenging mitochondrial superoxide could prevent CL097 or extracellular ATP activation of the NLRP3 inflammasome, we administered the mitochondrial-targeted superoxide dismutase mimetic MitoTEMPO. We also administered SEQEL1 (S1) or SEQEL3 (S3), which can suppress mitochondrial complex I-or III-generated superoxide production, respectively 47,48 . MitoTEMPO, S1 and S3 did not prevent CL097 or extracellular ATP activation of NLRP3 inflammasome (Fig. 7d-g). We used MitoTEMPO, S1 and S3 concentrations that do not inhibit OCR and have previously shown efficacy in other cell systems [47][48][49][50] . Finally, we tested whether increasing ROS production could rescue secreted IL-1β levels   in NDI1-expressing LPS-primed BMDMs treated with CL097. Paraquat-a known generator of superoxide production 51 -failed to increase secreted IL-1β levels (Fig. 7h). Collectively, our data indicate that CL097 requires inhibition of mitochondrial complex I to trigger NLRP3 inflammasome activation through an unidentified mitochondria-dependent mechanism (Fig. 7i).

Discussion
Our studies on LPS-primed BMDMs activated with extracellular ATP or CL097 have revealed three important aspects of mitochondrial ETC in controlling NLRP3 inflammasome activation. First, LPS priming increases mitochondrial ATP-dependent PCr levels that sustain NLRP3 inflammasome activation by both extracellular ATP and CL097. Importantly, mitochondrial ETC inhibitors maximally activate glycolysis, which is not able to sustain NLRP3 inflammasome activation. Second, mitochondrial ETC inhibitors are not sufficient to trigger NLRP3 inflammasome activation in LPS-primed BMDMs, consistent with previous findings 3 . Nevertheless, CL097 inhibits mitochondrial complex I to activate NLRP3 inflammasome in LPS-primed BMDMs, suggesting that CL097 targets mitochondrial complex I and some other unknown target(s) to activate the NLRP3 inflammasome. Moreover, the mechanism by which CL097 inhibition of mitochondrial complex I is necessary for NLRP3 inflammasome activation is not clear. Third, we find no evidence that mitochondrial ROS are necessary for NLRP3 inflammasome activation by extracellular ATP or CL097, although we cannot exclude nonmitochondrial ROS sources as potential inputs into NLRP3 activation. Collectively, our studies establish the necessity of the ETC to sustain NLRP3 inflammasome activation by both K + efflux-dependent, that is, extracellular ATP, and K + efflux-independent, that is, CL097, stimuli. It is important to note that we examined here only one critical aspect of inflammation-the production of IL-1β by the canonical NLRP3 inflammasome. Other cytokines that are linked to mitochondrial ETC function, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, may depend on mitochondrially generated ROS [52][53][54] . Mitochondrial DNA (mtDNA), known to activate the cGAS-STING pathway for induction of type I interferons, is potentially another input into NLRP3 inflammasome activation 55,56 . Our studies do not address or refute this mechanism. However, it is important to note that experimental strategies that deplete mtDNA also disable ETC function and thereby diminish mitochondrial ATP production. Thus, it is possible that depletion of mtDNA by TFAM ablation or cytidine monophosphate kinase 2 impairs NLRP3 inflammasome activation 55,57 , in part due to diminished mitochondrial ATP.
Our present studies do not address whether the mechanism described here would apply to stimuli, such as serum amyloid, that require longer period (24 h) of exposure 58,59 . Furthermore, we did not address whether ETC is necessary for the activation of other inflammasomes. Nevertheless, the genetic tools used in this study could be helpful in elucidating the necessity of mitochondrial ETC for different NLRP3 inflammasome stimuli as well as other distinct inflammasomes, such as AIM2 and NLRC4.

Methods
Mice. Male and female mice were used at 8-14 weeks of age. Littermate controls were used for all experiments. WT C57BL/6J mice were obtained from Jackson Laboratories and bred inhouse at Northwestern University. We used a previously published transgenic mouse line, which contains a lox-stop-lox-NDI1 targeting construct in the Rosa26 locus 26 . To generate mice that express NDI1 in hematopoietic cells, we bred mixed C57Bl/6J/N Rosa26 NDI1-lsl/wt mice with B6.Cg-Commd10 Tg(Vav1-icre)A2Kio /J mice from Jackson Laboratories to generate the conditional NDI1 transgenic mouse. Mice expressing AOX in myeloid cells were generated by breeding B6.129P2-Lyz2 tm1(cre)Ifo /J mice from Jackson Laboratories with previously published C57Bl/6N Rosa26 AOX-lsl/wt mice given to us by M. Szibor 28 . Previously published QPC fl/fl mice are C57Bl/6J background. All mouse lines were maintained at Northwestern University under specific pathogen-free conditions in ventilated microisolator cages with automatic water access. Teklad LM-485 mouse/ rat sterilizable diet chow (Envigo, catalog no. 7912) was provided ad libitum. Housing rooms had standard 12-h light/dark cycles and an ambient temperature of 23 °C. We complied with all relevant ethical regulations in accordance with Federal and University guidelines and protocols approved by IACUC and Northwestern University, protocol number IS00014481.
Oxygen consumption rate. The OCR was measured in a XF96 extracellular flux analyzer (Agilent Bioscience). BMDMs were plated at 0.15 × 10 6 cells per well of a XF96 plate, allowed to adhere overnight. Pretreated cells were treated with myxothiazol, piericidin A or oligomycin for 30 min, or dimethyl malonate for 3 h before OCR measurement. LPS-treated cells were primed with 100 ng ml -1 LPS for 6 h. At 1 h before OCR measurement, the medium was exchanged for Seahorse base RPMI (Agilent, catalog no. 103335-100, supplemented with glucose, methyl pyruvate, glutamine and uridine) in the presence or absence of 20 µg ml -1 VX-765 (Invivogen, catalog no. inh-vx765i-5) before initiation of the assay. Injection of ETC inhibitors and inflammasome activators occurred at the timepoints indicated in the figures. Final concentrations of drugs are included in the figure legends. Basal OCR was assessed by subtracting nonmitochondrial oxygen consumption (measured in the presence of 1 μM antimycin A (Sigma, catalog no. A8674) and 1 μM piericidin A) from the baseline OCR. Coupled OCR was assessed as the difference between basal OCR and OCR after addition of oligomycin.
NAD + :NADH ratio. BMDMs were plated at 0.15 × 10 6 cells per well in 96-well plates and allowed to adhere overnight. The NAD + /NADH ratio was measured in BMDMs treated with or without LPS for 4 h after pretreatment with or without of piericidin A or dimethyl malonate using the Promega NAD + /NADH Glo-Kit (Promega, catalog no. G9071) according to the manufacturer's instructions.
To avoid oxidation of cyclocreatine, we avoided repeated freeze-thaws of the powder and solution. Cell supernatant was collected and used for IL-1β and TNFα measurement via enzyme-linked immunosorbent assay (ELISA) (R&D Duoset, catalog nos. DY401-05 and DY410-05). ELISA kits were used according to manufacturer's instructions.

H 2 O 2 measurement in permeabilized BMDMs.
BMDMs were centrifuged at 500g and washed first with KHEB buffer (120 mM KCL, 5 mM HEPES, 1 mM EGTA, 0.3% BSA, pH 7.4 with KOH), followed by a wash with KHEB + 100 µg ml -1 saponin (Sigma, catalog no. 47036). A total of 150,000 cells were incubated in 96-well, clear-bottom plates (Corning, catalog no. 3603) in 50 µl KHEB + saponin for 15 min at room temperature. Following incubation, an additional 50 µl of KHEB buffer was added to each well along with 100 µl superoxide sensing solution (1.5 U ml -1 HRP (ThermoFisher), 25 KU ml -1 superoxide dismutase (Sigma, catalog no. S5395) and 25 μM Amplex Red (Invitrogen, catalog no. A222188)) was added to each well. Additional treatments were added to superoxide sensing solution as follows: 500 µM l-succinate (Sigma, catalog no. 224731), 500 nM piericidin A. Immediately following addition of superoxide sensing solution, the plate was placed in a SpectraMax M2 (Molecular Devices) plate reader set to mix plate and take readings every minute with excitation (544 nm) and emission (590 nm). For analysis, the fluorescent readings from the linear range of the reaction (15-30 min) were used to calculate the slope (RFU min -1 ). RFU min -1 readings from the BMDMs treated with superoxide sensing solution alone are subtracted as background readings.
ATP assay. A total of 2 million BMDMs were plated in a 12-well plate, as indicated above, and allowed to adhere overnight. BMDMs were treated with 100 nM piericidin A or 10 mM cyclocreatine for 30 min before the addition of 100 ng ml -1 LPS for 4 h where indicated. Nigericin (20 μM) was added where indicated for 20 min to allow for the initiation of NLRP3 inflammasome activation before cell death. Cells were harvested with ATP assay buffer (ATP Assay kit (Colorimetric/ Fluorometric), Abcam, catalog no. ab83355) and centrifuged at 14,000g for 5 min. The assay was performed according to the manufacturer's instructions using the fluorometric protocol. Fluorescent readings from each treated sample replicate are shown as relative to the fluorescent reading of the corresponding untreated sample.
Metabolomics. Two million BMDMs were allowed to adhere overnight in 12-well plates. The cells were treated with 50 nM oligomycin, 500 nM piericidin A, 100 nM myxothiazol or 10 mM cyclocreatine for 30 min, or 10 mM dimethyl malonate for 3 h, before stimulation with 100 ng ml -1 LPS for 4 h. To extract metabolites, 1 ml HPLC-grade methanol in water (80/20, v/v) cooled to -80 °C. Cells went through three complete freeze-thaw cycles in liquid nitrogen and a 37 °C waterbath before high-speed centrifugation at 4 °C. The supernatants, which contained metabolites, were collected and stored at -80 °C. The supernatants were dried in a SpeedVac concentrator (Thermo Savant). The dried metabolites were reconstituted in acetonitrile in analytical-grade water (50/50, v/v) and centrifuged to remove debris. A 10 μl aliquot of the sample was used for high-resolution HPLC-tandem mass spectrometry. High-resolution HPLC-tandem mass spectrometry was performed on a Q-Exactive (ThermoFisher Scientific) in line with an electrospray source and an UltiMate 3000 (ThermoFisher Scientific) series HPLC consisting of a binary pump, degasser and autosampler outfitted with a XBridge Amide column (Waters; 4.6 mm × 100 mm dimension and a 3.5 μm particle size). Mobile phase A contained water and acetonitrile (95/5, v/v), 10 mM ammonium hydroxide and 10 mM ammonium acetate (pH 9.0). Mobile phase B was 100% acetonitrile. The gradient was set to 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A, with a flow rate of 400 μl min -1 . The capillary of the electrospray ionization source was set to 275 °C, with sheath gas at 45 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. A mass/charge ratio scan ranging from 70 to 850 was used in positive/ negative polarity switching mode. MS1 data were collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 10 6 , with a maximum injection time of 200 ms. The top five precursor ions were fragmented using the higher-energy collisional dissociation cell with normalized collision energy of 30% in MS2 at a resolution of 17,500. Data were acquired with Xcalibur software (v.4.1; ThermoFisher Scientific). The resulting data were analyzed using MetaboAnalyst (v.4.0), normalized by total ion current. Significantly different metabolites between treatment groups were identified by one-way analysis of variance (ANOVA) with Fisher's least significant difference (LSD) post hoc analysis and then plotted as a heatmap. Peak areas of individual metabolites (that is, succinate, PCr, creatine) were graphed as arbitrary units and subjected to one-way ANOVA with Tukey t-test post hoc analysis for multiple comparisons.
RNA sequencing. BMDMs from WT and NDI1 mice were seeded in 12-well plates as described above. BMDMs were pretreated with or without 500 nM piericidin A for 30 min before addition of 100 ng ml -1 ultrapure LPS for 4 h. Samples were lysed with RLT Plus buffer (Qiagen, catalog no. 74134) with β-mercaptoethanol (1%) and homogenized with QIAshredder Spin Columns (Qiagen, catalog no. 79654). RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, catalog no. 74134), according to the manufacturer's protocol plus on-column DNase treatment using the RNase-Free DNase Set (Qiagen, catalog no. 79254). RNA was quantified and quality control performed using the Agilent 4200 TapeStation RNA ScreenTape. mRNA libraries were prepared using NEBNext Ultra Kit with polyA selection (New England BioLabs). Sequencing of libraries was performed using a Next-Seq 500 High output for 75 cycles (Illumina). Raw BCL read files were demultiplexed and FASTQ files were generated using bcl2fastq and trimmed using Trimmomatic 60 . The reads were then aligned to the mouse mm10 reference genome using STAR to generate BAM files 61 . HTSeq was used to count reads in the exons of genes 62 . Likelihood ratio tests for all samples and all detected transcripts and pairwise differential gene expression analyses were carried out using the R package DESeq2 (ref. 63 ).

LPS induction of IL-1β protein in mice.
Crude O5:B55 LPS (Sigma, catalog no. L2880) was prepared at 5 mg ml -1 in PBS. Littermate mice (WT, QPC-KO, QPC-KO/AOX and NDI1) were administered at 100 mg kg -1 or 50 mg kg -1 LPS, as specified in figure legends, via the intraperitoneal (i.p.) route for 2 h. For cyclocreatine treatments, cyclocreatine was made freshly before each experiment at 10 mg ml -1 in PBS and brought to a pH of 7.4. To avoid oxidation of cyclocreatine powder, we limited repeated freeze-thaws and exposure of individual vials to air. Cyclocreatine in the drinking water was prepared at 1% weight/volume and administered overnight ad libitum. Cyclocreatine solution or PBS vehicle was administered i.p. to C57Bl/6J mice at 400 mg kg -1 for 2 h before administration of 50 mg kg -1 crude LPS for 2 h. Whole blood samples were harvested via retro-orbital bleed before euthanasia in a CO 2 chamber. Samples were allowed to clot and then centrifuged at 14,000g for 15 min and serum was collected. IL-1β concentration in serum was measured using the IL-1β Quantikine ELISA kit (R&D Systems, catalog no. MLB00C) as per the manufacturer's instructions.
Cell death assay using LDH release. BMDMs were plated at 0.15 × 10 6 in a 96-well plate and allowed to adhere overnight. Cells were primed with LPS (100 ng ml -1 ) and the NLRP3 inflammasome activated with Nigericin (20 μM) or CL097 (70 μM) for the times indicated with or without the caspase-1 inhibitor VX-765 (20 μg ml -1 ). Plates were spun down at 500g for 1 min and cell culture supernatant was transferred to a fresh plate. Assay was performed on supernatant according to manufacturer's instruction using the Cytotoxicity Detection Kit (LDH) (Sigma, catalog no. 11644793001).
Membrane potential measurement. BMDMs were plated at 2 million cells per well in a 12-well plate and allowed to adhere overnight. Cells were primed for 4 h with LPS (100 ng ml -1 ) in the presence or absence of FCCP (10 μM), piericidin A (Pier) (100 nM), Oligomycin (50 nM) or Myxothiazol (100 nM). TMRE (Abcam, catalog no. ab113852) was added at a concentration of 200 nM for 30 min. Cells were washed with PBS and removed from the plate with Accutase (Fisher Scientific, catalog no. NC9839010) before resuspension in PBS supplemented with 10% NU-Serum IV (Fisher Scientific, catalog no. CB-55004). Data were obtained using a BD FACSymphony A5-Laser Anaylzer (BD Biosciences).
Statistical analysis. Statistical analyses were performed in GraphPad Prism v.9 software using statistical tests indicated in the figure legends. Statistical analyses of metabolomics data were performed using Metaboanalyst 64 . Data are presented as mean ± s.e.m. with a minimum of n = 3 independent experiments, except Fig. 6b-g, which are presented as mean ± s.d. of four technical replicates. Specific number of replicates are indicated in figure legends. Experiments were neither randomized nor blinded. Statistical significance was determined by a two-tailed t-test, a one-sample t-test, an ANOVA followed by Tukey's multiple comparison test or an ANOVA followed by Fisher's LSD. Specific tests are indicated in figure legends. Statistical significance was defined as follows: P < 0.05. Data distribution was assumed to be normal, but this was not tested formally. No statistical methods were used to predetermine sample sizes, but our samples sizes are similar to those reported in previous publications 19,34,41 . Plated cells were allocated randomly to each treatment group; C57Bl/6 mice were assigned randomly to each treatment group. Data collection and analysis were not performed blind to the conditions of the experiments. Experiments were excluded from analysis if the controls did not work; data from successfully completed experiments were not excluded. Fig. 1 | Dimethyl malonate (DMM) inhibits mitochondrial complex II. a) Timeline of treatment of BMDMs with metabolic inhibitors, LPS, and inflammasome activators. b) Schematic of the mitochondrial ETC, indicating forward and reverse (RET) electron transport. Dimethyl malonate (DMM) inhibits mitochondrial complex II, preventing succinate oxidation and linked electron transport in either direction. c) OCR in BMDMs after 3 hours treatment with or without 10 mM DMM (Untreated: N = 11; 10 mM DMM: N = 6). d) Heatmap of significantly altered metabolites in BMDMs treated with DMM (10 mM) with or without LPS (100 ng/mL) for 4 hours. The relative abundance of each metabolite is depicted as z score across rows (red, high; blue, low). (N = 5, each treatment). e) Succinate concentration (AU, arbitrary units) in WT BMDMs with or without treatment with LPS (100 ng/mL) and DMM (10 mM) for 4 h (N = 5, each treatment). f) NAD + /NADH ratio in BMDMs after 4 hours treatment with or without LPS (100 ng/mL), with or without DMM (10 mM) (N = 4, each treatment). Data are means + /− SEM. * p < 0.05, two-tailed t-test (c *p < 0.0001), one-way ANOVA with Tukey test for multiple comparisons (e *p = 0.0057 UT/DMM, *p = 0.0205 LPS/LPS + DMM), or one-way analysis of variance (ANOVA) with Fisher's LSD (d). N indicates number of individual mice. Parts of this figure were created with BioRender.com. Fig. 3 | Piericidin A inhibits mitochondrial complex I. a) Schematic of the mitochondrial ETC, indicating forward and reverse (RET) electron transport. Piericidin A inhibits mitochondrial complex I, preventing proton pumping, superoxide production, and both forward and reverse electron transport. b) OCR in BMDMs after 1-hour treatment with or without 100 nM or 500 nM piericidin A (N = 8 basal; N = 4 100 nM; N = 6 500 nM). c) NAD + /NADH ratio in BMDMs after 4-hour treatment with or without LPS (100 ng/mL), with or without piericidin A (500 nM) (N = 4 for each condition). d) Heatmap of significantly altered metabolites in BMDMs treated with piericidin A (500 nM) with or without LPS (100 ng/mL) for 4 hours. The relative abundance of each metabolite is depicted as z score across rows (red, high; blue, low). (N = 5 for each condition). e) Succinate concentration (AU, arbitrary units) in WT BMDMs with or without treatment with LPS (100 ng/mL), with or without piericidin A (500 nM) for 4 hours (N = 5, for each condition). Data are means + /− SEM. * p < 0.05, one-way ANOVA with Tukey test for multiple comparisons (b *p < 0.0001; c *p = 0.0442 UT/Piericidin A, *p = 0.0426 LPS/LPS + Piericidin A; e *p = 0.0258 UT/LPS, *p = 0.0043 LPS/LPS + Piericidin), or one-way ANOVA with Fisher's LSD (d). Parts of this figure were created with BioRender.com. Fig. 7 | oligomycin or FCCP cause an increase or decrease in mitochondrial membrane potential, respectively. a) Schematic of mitochondrial membrane potential (Ψψm) at baseline (top), during in the presence of oligomycin (middle), and in the presence of FCCP (bottom). At baseline, mitochondrial complexes I, III and IV pump protons across the inner mitochondrial membrane to generate and maintain a high membrane potential. Mitochondrial complex V uses this proton motive force to generate ATP from ADP and Pi . Oligomycin inhibits mitochondrial complex V, preventing the passage of protons through complex V into the mitochondrial matrix. This causes an increase in the membrane potential as protons build up in the intermembrane space. FCCP is a protonophore and allows for the free passage of protons across the inner mitochondrial membrane. This decreases the membrane potential, preventing ATP generation. b) Relative MFI (geometric mean of TMRE stain, relative to UT control) of BMDMs treated with LPS (100 ng/mL), or not, with FCCP (10μM), piericidin A (Pier) (100 nM), Oligomycin (50 nM), or Myxothiazol (100 nM) (N = 5 Myxo, FCCP + LPS, Myxo + LPS; N = 7 FCCP, Pier, Pier + LPS; N = 9 Oligo, Oligo + LPS, UT). c) Example gating strategy for b with representative histograms of untreated, FCCP treated, and oligomycin treated BMDMs. Cell counts are standardized to mode. Data are means + /− SEM. * p < 0.05, one-sample t-test (b *p < 0.0001 UT/FCCP, p = 0.0116 UT/Oligo). Parts of this figure were created with BioRender.com.